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Bio 108 - 3/6/2000 Morphogenetic Movements (contd) • Contact information – Bruce Blumberg – Office 4216 BioSci II • office hours W 3-4 F 12-1 (or by appointment in exceptional cases) – phone 824-8573 – [email protected] (preferred contact mode) • You are responsible for what I lecture about and what is covered in the assigned reading in Alberts, et al. – Generally speaking, I will stick close to the book as did the other lecturers • Today – morphogenetic movements • neurulation • neural crest migration – cell determination BioSci 108 lecture 23 (Blumberg) page 1 ©copyright Bruce Blumberg 2000. All rights reserved Neurulation • • • Last time we finished with gastrulation The next developmental step is when the gastrula ectoderm is induced to form neural tissue by the underlying mesoderm and notochord (Figure 21-10) – neural plate thickens and rolls up into a tube – neural tube pinches off from the rest of the cell sheet – the inducing mesoderm can lead to the formation of an ectopic neural tube if transplanted to a new embryo, much like the organizer transplant – process is driven by changes in cell shape (21-11) • cells become columnar • apical actin bundles contract and cause curling of neural plate subsequently, dorsal cells of the neural tube pinch off and migrate out through the mesoderm – these neural crest cells will contribute to numerous structures and tissues • peripheral nervous system • sympathetic ganglia • Schwann cells • pigment cells – head neural crest will differentiate into cartilage, bone and connective tissue • these typically derive from mesoderm in other parts of the body BioSci 108 lecture 23 (Blumberg) page 2 ©copyright Bruce Blumberg 2000. All rights reserved Cell adhesion • Changes in cell adhesion molecules are important for morphogenetic movements – classic experiments showed that cells can recognize others of the same type (figure 21-14) • experiment: – dissociate gastrula cells – allow to reassociate • observation: – cells sort out according to tissue type – outer epidermis – central neural tube – mesoderm in between • inference: – cells can identify each other – basis of identification is cell surface molecules called cadherins (21-15) • Ca-dependent glycoproteins that are differentially expressed in the early embryo • cadherins control the formation and dissolution of cell sheets AND • provide an anchor for cytoskeleton within the cells • recognition of cadherin types and combinations mediates interactions between cells and their neighbors. BioSci 108 lecture 23 (Blumberg) page 3 ©copyright Bruce Blumberg 2000. All rights reserved Cells and the extracellular matrix • In addition to adhering to each other, cells need to interact with the extracellular matrix – mediated through another class of cell surface proteins called integrins – integrins serve as transmembrane linkers between the ECM and cytoskeleton – How to test the role of this interaction? • experiment: – block cells from binding to fibronectin with a synthetic peptide that saturates the binding site • observation: – cells do not migrate and appear only loosely adherent • conclusion – cell interaction with integrins is very important for migration – Mutations in integrins lead to embryonic patterning defects • Drosophila mutation lethal (1) myospheroid leads to failure of muscle attachment – embryos burst at about 24 hours after fertilization when the first muscular movements take place – mutation is in an integrin subunit found in muscles BioSci 108 lecture 23 (Blumberg) page 4 ©copyright Bruce Blumberg 2000. All rights reserved Invasion and migration • • • Many types of cells migrate in the early embryo from their point of origin – muscle cells – neural crest – blood cells – primordial germ cells – neurons how to follow migrations? – Mark cells at the beginning of their journey and follow them • nontoxic dye • heritable genetic marker – quail/chicken chimeras Neural crest follows distinct migratory pathways (21-17) – much of this work was done by Marianne BronnerFraser who was previously at UCI – migrating cells extend cellular processes that “test” for interactions with neighboring cells • depending on the types of interactions found, the cells go one direction or another • cell surface receptors again mediate this process – some types attract or promote adhesion – others inhibit movement and repel connections – Corey Goodman and neural cell migration BioSci 108 lecture 23 (Blumberg) page 5 ©copyright Bruce Blumberg 2000. All rights reserved Cell Diversification in the early embryo • • All of the information needed to form any type of organism is contained in a single cell – the central goal of developmental biology is to understand how • what are the molecules? • how do they interact? • what are the physical processes? Question - does the genome remain intact and totipotent during development or do cells become specialized by genomic modifications? – Answer seems obvious but an important early developmental model was the worm Ascaris. • Significant parts of the genome are lost during development and differentiation – experiment: (Figure 21-20) • transplant the nucleus from a differentiated cell from adult frog to egg lacking nucleus • what is the developmental potential of the resulting embryo? – Observation: • normal tadpoles developed that had characteristics of the transplanted nucleus (albino) – conclusion: • genome remains intact during development • cells differ because they express different genes BioSci 108 lecture 23 (Blumberg) page 6 ©copyright Bruce Blumberg 2000. All rights reserved Cell diversification (contd) • – Resulting animals are clones, exact copies of the donor animal – Such experiments were previously difficult and not so reproducible – today, the technology has advanced sufficiently that mammals can be readily cloned • for $250,000 you can have your pet cloned! • it is only a matter of time before someone does this with humans Cytoplasmic determinants – in most plants and animals, the egg is chemically asymmetrical • certain components are concentrated in specific regions • these localized cytoplasmic determinants vary in importance between species – development was previously characterized in two broad ways • in mosaic development, if a blastomere is removed, the structures derived from it fail to form e.g. sea urchin – Inference is that determinants are important • in regulative development the embryo develops normally despite blastomere removal – determinants are not important – no animals are completely either way! BioSci 108 lecture 23 (Blumberg) page 7 ©copyright Bruce Blumberg 2000. All rights reserved Embryonic induction • • Induction - the switching of cells from one pathway into another by the influence of an adjacent group of cells – one of the most important processes during early development of virtually all organisms – experiments that demonstrated induction are some of the most famous experiments in biology Organizer transplant (Hans Spemann) – until recently, the only Nobel Prize awarded for Developmental Biology – experiment: • transplant the dorsal lip of the blastopore to the ventral side of a host embryo – observation: • a complete secondary axis was formed from both host and graft tissue – inference: • the transplant was able to organize the host tissue into a secondary axis • factor(s) from the graft were able to do this – over the years, many such experiments were done until it became clear that many, many types of molecules could induce secondary axes in salamanders • put field into disrepute for many years • turns out that salamander is promiscuous for neural induction BioSci 108 lecture 23 (Blumberg) page 8 ©copyright Bruce Blumberg 2000. All rights reserved Embryonic induction (contd) • • Xenopus saved the day – unlike salamander mesoderm and neural tissue could only be induced in Xenopus by a few factors – Pieter Niewukoop performed a classic induction experiment (figure 21-21) • isolated animal pole and vegetal pole cells • if cultured in isolation, the animal pole cells formed epidermis and the vegetal pole cells stayed as they were. • But when put together, mesoderm was formed! • This simple mesoderm induction assay was the basis for nearly all of the advances in molecular embryology in recent years – Tiedemanns and Grunz spent many years purifying factors from embryos that could induce mesoderm • these turned out to be growth factors in the FGF and TGF-beta families Later, a number of laboratories began to study the molecular nature of the organizer – Viktor Hamburger - Hans Spemann and the Organizer, The Heritage of Experimental Embryology – Prompted by this book, professor Cho and I constructed the first cDNA library from the dorsal lip and identified many factors that were important for its properties (Science 253, 194-196. BioSci 108 lecture 23 (Blumberg) page 9 ©copyright Bruce Blumberg 2000. All rights reserved Embryonic induction (contd) • • • Sequential induction is responsible for much embryonic patterning (Fig 21-22) – a series of inductive interactions can generate many kinds of cells, starting from only a few Inductive abilities of cells differs – although all vegetal cells can induce mesoderm, only cells from the dorsal side can induce dorsal mesoderm (organizer) (Fig 21-23) – There are at least three different signals ( and probably many more) responsible for mesoderm induction in the early Xenopus embryo – fertilization -> cortical rotation – cortical rotation -> asymmetrical distribution of dorsal determining factors (several possibilities) • dorsal and ventral endoderm – mesoderm and organizer induced by signal from vegetal cells (signals 1 and 2) – organizer dorsalizes adjacent mesoderm (signal 3) mesoderm induction involves a variety of signaling molecules – TGF-beta family (activin, Vg-1, BMP2,4) – Wnt family – FGF family (FGF4) – inhibitors of above • chordin, noggin, cerberus BioSci 108 lecture 23 (Blumberg) page 10 ©copyright Bruce Blumberg 2000. All rights reserved Actions of inducing molecules • • • • One model for how the organizer is induced – cortical rotation leads to activation of Vg-1 on the dorsal side of the embryo – Vg-1 protein in vegetal cells induces the organizer – attractive model but not completely correct • many so-called dorsalizing signals are, in fact, inhibitors of ventral mesoderm and ectoderm induction Location (Fig 21-26) – can be intracellular or extracellular – molecules can act at both short and long range nature – secreted molecules – bound to cell surface secreted signals are called morphogens – morphogens are diffusible substances that pattern the embryo – in its strictest definition, a morphogen patterns tissues depending on its concentration • model originated with Alan Turing a British mathemetician who almost single-handedly finished off the Germans in WWII – he solved the famous “enigma” code that allowed the Allies to decipher German military transmissions BioSci 108 lecture 23 (Blumberg) page 11 ©copyright Bruce Blumberg 2000. All rights reserved Morphogens and gradients • – Gradient model was popularized by Child, Huxley and De Beer • essential feature is that activity gradients led to the formation of specialized regions of the embryo – Louis Wolpert provided many important theoretical treatments, observed that positional signaling occurs over small distances, -.1-2 mm – Francis Crick provided legitimacy to the model with a quantitative treatment of the activity of diffusible substances how do morphogens work (Fig 21-26) – essential feature is that morphogens diffuse from a source and are degraded or captured by a sink • if there was no loss or destruction of the morphogen there would ultimately be no difference in the concentration – cells sense their position in a morphogen gradient and respond according to the concentration of morphogen they encounter • ectodermal cells exposed to little or no activin become epidermis • low levels of activin induce muscle • higher levels induce notochord BioSci 108 lecture 23 (Blumberg) page 12 ©copyright Bruce Blumberg 2000. 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